A high-throughput screen to identify enhancers of ADAR-mediated RNA-editing

• Published in: RNA biology Vol. 10; no. 2; pp. 192 - 204
• Main Authors: Garncarz, Wojciech, Tariq, Aamira, Handl, Cornelia, Pusch, Oliver, Jantsch, Michael F.
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 01.02.2013; Landes Bioscience
• Subjects: 3' Untranslated Regions; Adenosine Deaminase - genetics; Adenosine Deaminase - metabolism; Animals; DNA, Complementary - genetics; DNA, Complementary - metabolism; DSS1; Enhancer Elements, Genetic; Genetic Vectors - genetics; Genetic Vectors - metabolism; HEK293 Cells; HeLa Cells; Heterogeneous-Nuclear Ribonucleoprotein Group A-B - genetics; Heterogeneous-Nuclear Ribonucleoprotein Group A-B - metabolism; High-Throughput Nucleotide Sequencing - methods; Humans; Proteasome Endopeptidase Complex - genetics; Proteasome Endopeptidase Complex - metabolism; Rats; regulation of RNA-editing; RNA Editing; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA-binding; RNA-Binding Proteins; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Technical Paper; TREX-2; yeast screen; DSS1; TREX-2; RNA-binding; RNA-editing; yeast screen; regulation of RNA-editing
• ISSN: 1547-6286; 1555-8584
• DOI: 10.4161/rna.23208

Adenosine to inosine deamination of RNA is widespread in metazoa. Inosines are recognized as guanosines and, therefore, this RNA-editing can influence the coding potential, localization and stability of RNAs. Therefore, RNA editing contributes to the diversification of the transcriptome in a flexible manner. The editing reaction is performed by adenosine deaminases that act on RNA (ADARs), which are essential for normal life and development in many organisms. Changes in editing levels are observed during development but also in neurological pathologies like schizophrenia, depression or tumors. Frequently, changes in editing levels are not reflected by changes in ADAR levels suggesting a regulation of enzyme activity. Until now, only a few factors are known that influence the activity of ADARs. Here we present a two-stage in vivo editing screen aimed to isolate enhancers of editing. A primary, high-throughput yeast-screen is combined with a more accurate secondary screen in mammalian cells that uses a fluorescent read-out to detect minor differences in RNA-editing. The screen was successfully employed to identify DSS1/SHFM1, the RNA binding protein hnRNP A2/B1 and a 3′ UTR as enhancers of editing. By varying intracellular DSS1/SHFM1 levels, we can modulate A to I editing by up to 30%. Proteomic analysis indicates an interaction of DSS1/SHFM1 and hnRNP A2/B1 suggesting that both factors may act by altering the cellular RNP landscape. An extension of this screen to cDNAs from different tissues or developmental stages may prove useful for the identification of additional enhancers of RNA-editing.